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International Journal of Applied Research and Technology

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Esxon Publishers International Journal of Applied Research and Technology ISSN 2277-0585 Publication details, including instructions for authors and subscription information: http://www.esxpublishers.com

Comparison of Phosphoric and Nitric Acid Digestion Procedures for the Determination of Metals in Cow Parts by Atomic Absorption Spectrophotometry Nwude, D. O., Babayemi, J. O. and Owonifari, W. Bells University of Technology, Ota, Nigeria

Available online: December 24, 2012

To cite this article: Nwude, D. O., Babayemi, J. O. and Owonifari, W. (2012). Comparison of Phosphoric and Nitric Acid Digestion Procedures for the Determination of Metals in Cow Parts by Atomic Absorption Spectrophotometry. International Journal of Applied Research and Technology. 1(8): 100 – 106.

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International Journal of Applied Research and Technology Vol. 1, No. 8, December 2012. 100 – 106.

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Comparison of Phosphoric and Nitric Acid Digestion Procedures for the Determination of Metals in Cow Parts by Atomic Absorption Spectrophotometry Nwude, D. O., Babayemi, J. O. and Owonifari, W. Bells University of Technology, Ota, Nigeria

(Received: 28 November 2012 / Accepted: 08 December 2012 / Published: 24 December 2012)

Abstract The efficiency and reliability of the use of phosphoric acid (H3PO4) in the digestion of animal samples for metal analysis was assessed in this study. The results obtained from the analysis of metals in animal parts after digesting the samples with H3PO4 have been reported to be comparable with those reported for similar studies elsewhere where digestion was accomplished using nitric acid (HNO3). However, there have not been any studies to assess the efficiency of using H 3PO4 for digestion compared to HNO3. This study therefore undertakes to assess the efficiency of using H3PO4 for digestion of animal parts, compared to HNO3, by using the two mineral acids differently in the digestion of 15 animal samples consisting of kidney, liver and muscle from randomly sampled 5 cows at slaughter at Atan Abattoir, Ota, Nigeria, to determine the levels of some metals. After digestion, atomic absorption spectrophotometer was used for the analysis of the metals: Arsenic (As), Nickel (Ni), Cobalt (Co), Lead (Pb) and Cadmium (Cd). There were variations in the colour of filtrates of the digested samples obtained. For digestion with HNO 3, the mean concentrations of metals ranged from 4.5235.951 mg/kg, As (standard deviation: 0.705-1.656); 0.054-0.108 mg/kg, Ni (standard deviation: 0.058-0.083); 0.010-0.029 mg/kg, Co (standard deviation: 0.020-0.042); 0.620-0.721 mg/kg, Pb (standard deviation: 0.098-0.379); and 0.057-0.086 mg/kg, Cd (standard deviation: 0.012-0.031). For digestion with H3PO4, the mean levels ranged from 5.486-6.843 mg/kg, As (standard deviation: 2.280-3.334); Co was not detected; 0.133-0.143 mg/kg, Cd (standard deviation: 0.007-0.037); 0.012-0.039 mg/kg, Ni (standard deviation: 0.020-0.037); and 0.476-0.638 mg/kg, Pb (standard deviation: 0.195-0.200). It may be concluded that though the digestion with HNO3 yielded slightly higher levels of some of the metals than did the H3PO4, statistically the results were not significantly different, except for Cd. Keywords: Nitric Acid, Phosphoric Acid, Digestion Procedure, Cow Parts, Metals

Corresponding author: E-mail: [email protected] ISSN 2277-0585 © 2012 Esxon Publishers. All rights reserved.


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Introduction Previous studies have attempted to evaluate the efficiency of some digestion procedures for metals determination in biological samples (Gazi et al., 2009; Ashokaa et al., 2009; Moreiraa et al., 2005; Nemati et al., 2010; Bakircioglu et al., 2011; Bizzi et al., 2011; Saavedraa et al., 2004). Considering the cost of reagents and the desired level of accuracies in chemical analysis, some objectives of previous works tend towards reducing both the reagent volumes and the variability of the residues as a result of the process of decomposition (Gonzalez et al., 2009). Aiming at achieving complete destruction of organic matrix of the biological samples, some researchers have used aqua regia in the digestion procedues (Olowu et al. 2010; Alinnor and Obiji, 2010; Koréneková, 2002), or a mixture of two or more of other mineral acids (Gbaruko and Friday, 2007); in other instances, hydrogen peroxide is introduced (Begum, 2009). According to a standard method for the determination of metals in wildlife tissues using flame atomic absorption spectrophotometry technique (Neugebauer, 2000), the use of concentrated HNO3 combined with heating may be adequate. Though H3PO4 is a weak mineral acid, it is of higher purity (85%) than the corresponding HNO 3 (69-72%). In the process of digestion (of animal samples) where 5 ml of concentrated HNO 3 may be required for about 1 g of the animal sample to be digested, about 2 ml of H3PO4 could perform the same task (Nwude et al., 2011), though the process could be more gradual than for HNO3, and the mixture must be heated for actual digestion to take place. It could then be inferred that in terms of purity and volume required for digestion, H 3PO4 is of higher advantage. The results obtained from the analysis of metals in animal parts after digesting the samples with H 3PO4 (Nwude et al., 2010a; 2010b; 2010c; 2011) have been reported to be comparable with those reported for similar studies elsewhere where digestion was accomplished using HNO3 or other conventional methods (Iwugbe 2008; Miranda et al., 2005; Blanco-Penedo et al., 2006) . However, there have not been any studies to assess the efficiency of using H 3PO4 for digestion compared to HNO3. This study therefore undertakes to assess the efficiency of using H3PO4 for digestion of animal parts, compared to HNO 3, by using the two mineral acids differently in the digestion of cow parts to determine the levels of some metals. Materials and Methods Fifteen samples comprising three cow parts (kidney, liver and muscle tissue) from five different cows were obtained fresh from an abattoir at Atan in Ota, Ogun state. Each part was divided into two, one to be digested with HNO 3 and the other with H3PO4. 2.5g of each part were placed in digestion test tubes previously washed and rinsed with distilled and deionized water. 2 ml concentrated HNO3 were added to one part and the same volume of concentrated H3PO4 to the other. For digestion with HNO3, this was allowed to take place at room temperature (30 oC) for 262 minutes; the time was then varied for some of the samples at 245 minutes. For digestion with H 3PO4, the test tubes with their contents were arranged on test tube rack and heated at 186 oC in a digestion chamber for 35 minutes; the temperature and time were then varied at 171 oC and 33 minutes. Then, 50 ml de-ionized water were added to the contents in each test-tube and thoroughly shaken. It was then filtered into 100 ml standard flask and made up to mark with de-ionized water. Analysis for metals was carried out using Atomic Absorption Spectrophotometer, S Series 712354 v1.27, at wavelengths of 240.7, 228.8, 193.7, 217.0 and 232.0 nm for Co, Cd, As, Pb and Ni respectively. Analysis of blanks was carried out to detect contaminations of reagents. Descriptive statistics were determined and paired data t-test was conducted to see if there were significant differences at 95% confidence level, using SPSS. Results and Discussion Table 1 shows variation in colour of filtrate of the digested samples as a function of the type of mineral acid used for digestion and duration of digestion. For digestion with HNO 3, concentrations of metals in the kidney (Table 2) ranged from 2.414-6.761 mg/kg, As; 0.013-0.219 mg/kg, Ni; ND-0.045 mg/kg, Co; 0.064-0.897 mg/kg, Pb; and 0.058-0.129 mg/kg, Cd; in the liver , the ranges were 4.812-6.619 mg/kg. As; 0.012-0.121 mg/kg, Ni; ND-0.058 mg/kg, Co; 0.511-0.767 mg/kg, Pb; and 0.038-0.068 mg/kg, Cd; in the muscle tissue, the levels ranged from 1.792-6.237 mg/kg, As; ND-0.130 mg/kg, Ni; ND-0.098 mg/kg, Co; 0.320-1.138 mg/kg, Pb; and 0.034-0.091 mg/kg, Cd. For digestion with H3PO4, the levels in the kidney ranged from 0.865-8.613 mg/kg, As; ND-0.055 mg/kg, Ni; Co was not detected; 0.362-0.775 mg/kg, Pb; and 0.092-0.194 mg/kg, Cd; in the liver, the levels were 4.653-10.190 mg/kg, As; ND-0.087 mg/kg, Ni; Co was not detected; 0.176-0.668 mg/kg, Pb; and 0.110-0.167 mg/kg, Cd. The levels in the muscle tissue were 0.939-10.133 mg/kg, As; 0.0190.065 mg/kg, Ni; Co was not detected; 0.318-0.811 mg/kg, Pb; and 0.127-0.143 mg/kg, Cd. The elements were not detected in the blank. For digestion with HNO3 (Table 3), the mean concentrations of metals ranged from 4.523-5.951 mg/kg, As (standard deviation: 0.705-1.656); 0.054-0.108 mg/kg, Ni (standard deviation: 0.058-0.083); 0.010-0.029 mg/kg, Co (standard deviation: 0.020-0.042); 0.620-0.721 mg/kg, Pb (standard deviation: 0.098-0.379); and 0.057-0.086 mg/kg, Cd (standard deviation: 0.012-0.031). For digestion with H3PO4, the mean levels ranged from 5.486-6.843 mg/kg, As (standard deviation: 2.280-3.334); Co was not detected; 0.133-0.143 mg/kg, Cd (standard deviation: 0.007-0.037); 0.0120.039 mg/kg, Ni (standard deviation: 0.020-0.037); and 0.476-0.638 mg/kg, Pb (standard deviation: 0.195-0.200). The elements were not detected in the blank. The colour of filtrate of the digest seemed to vary with temperature and digestion time. Digestion with HNO3 at longer time gave rise to light yellow filtrate of the digest; while at shorter time, some deep yellow filtrate was observed. Digestion with H3PO4 at higher temperature and longer time yielded filtrate with brown colour; but some deep brown colour was observed as the temperature and time were decreased. The yellow colour observed for digestion with HNO3 could indicate a better digestion than it was obtained for H 3PO4 which yielded brown


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filtrate. Since H3PO4 is a weak mineral acid, there is the possibility of the organic components of the samples not being completely destroyed; that might have given rise to the brown colour. Generally, the levels of As were the highest and those of Co being the least. Liver seemed to be the target organ for As. The levels of As observed in the liver both from digestion with HNO 3 and H3PO4 were the highest (Fig. 1a). The determination of As and Cd in the cow parts appeared to be favoured by digestion with H 3PO4 (Fig. 1a, e). As has been reported to be better preserved in aqueous solution with H3PO4 (As itself forming a triprotic acid as H3PO4) (Daus et al., 2006); perhaps this contributed to its better analysis using the latter digestion procedure. Further studies are needed to substantiate this. On the other way, H3PO4 method of digestion may not be appropriate for determination of Ni and Co in animal tissues. Since cobalt phosphate is insoluble in water (slightly soluble in cold water), perhaps this made it unavailable in solution for determination by atomic absorption spectrometry, and hence the non-detectable ranges observed. The levels of Ni were low compared with those observed for HNO 3 method of digestion (Fig. 1b), while Co was not detected when the samples were digested with H3PO4 (Fig. 1c). In the case of Pb (Fig. 1d), there seemed to be no selectivity for the method of digestion. Spectral and transport interferences have been suggested to be a main limitation in the determination of Pb in phosphoric acid medium; and the reason being that the analytical line for Pb at 217.0005nm overlaps with that of PO molecular absorption bands at 216.99nm in the air-acetylene flame (Raposo Jr et al., 2008). Though the determination of As in the samples seemed to be favoured by digestion with H 3PO4, a better precision was observed for HNO3 method of digestion (Table 3); but reverse seemed to be the case for the determination of Ni: the standard deviations were lower for HNO3 method of digestion. In the case of Pb, there was no conspicuous difference in the precisions. The precision in the determination of Cd using H 3PO4 seemed to be better than that for HNO3. The paired ttest was conducted at 95% confidence level (α = 0.05). The observed P-values were all greater than 0.05, except for Cd (Table 4). It may then be inferred that while the case for Cd seemed to be consistently an exception, there was no significant difference in the results obtained for the two methods. However, the exceptional case for Cd may suggest some unique reactions between H3PO4 and Cd in the animal tissues in the process of digestion, including the accompanying conditions. Further studies are suggested. Conclusion and Recommendations The findings shows that the determination of the levels of Cd in all the organs was favoured by digestion with H 3PO4, as the levels were consistently slightly higher than those observed for the digestion with HNO3. On the other way, H3PO4 may not be appropriate for digestion of animal samples for the determination of Co, as it was not detected, though the digestion with HNO3 yielded slightly higher levels of some of the metals than H3PO4, statistically the results were not significantly different, except for Cd. References Alinnor, IJ, Obiji IA (2010). Assessment of Trace Metal Composition in Fish Samples from Nworie River. Pakistan Journal of Nutrition 9 (1): 81-85, 2010. Ashokaa, S, Peakea BM, Bremner G, Hagemana KJ, Reid MR (2009). Comparison of digestion methods for ICP-MS determination of trace elements in fish tissues. Analytica Chimica Acta 653: 191–199. Bakircioglu, D, Kurtulus YB, Ucar G (2011). Determination of some traces metal levels in cheese samples packaged in plastic and tin containers by ICP-OES after dry, wet and microwave digestion. Food and Chemical Toxicology 49: 202–207. Begum, A. HariKrishna S, Khan I (2009). Analysis of Heavy metals in Water, Sediments and Fish samples of Madivala Lakes of Bangalore, Karnataka. International Journal of ChemTech Research 1(2): 245-249. CODEN( USA): IJCRGG ISSN : 0974-4290 Bizzi, CA, Flores EMM, Barin JS, Garcia EE, Nóbrega JA (2011). Understanding the process of microwave-assisted digestion combining diluted nitric acid and oxygen as auxiliary reagent. Microchemical Journal (in press). Blanco-Penedo I, Cruz JM, Lopez-alonso M, Miranda M, Castillo C et al. (2006). Influence of copper status on the accumulation of toxic and essential metals in cattle. Environment International 32: 901-906. Daus B, Weiss H, Mattusch J, Wennrich R (2006). Preservation of arsenic species in water samples using phosphoric acid – Limitations and long-term stability. Talanta 69: 430–434. Gbaruko BC, Friday OU (2007). Bioaccumulation of heavy metals in some fauna and flora. International Journal of Environmental Science and Technology 4 (2): 197-202, 2007 Gonzalez, MH, Souzaa GB, Oliveirac RV, Foratod LA, Nobregac JA, Nogueiraa ARA (2009). Microwave-assisted digestion procedures for biological samples with diluted nitric acid: Identification of reaction products. Talanta 79: 396–401. http://www.e-journals.net Iwugbe, CMA (2008). Heavy metal composition of livers and kidneys of cattle from southern Nigeria. Vet. Arch. 78: 401410. Kazi TG, Jamalia MK, Araina MB, Afridi HI, Jalbani N, Sarfraza RA, Ansari R (2009). Evaluation of an ultrasonic acid digestion procedure for total heavy metals determination in environmental and biological samples. Journal of Hazardous Materials 161: 1391–1398. Koréneková B, Skalická M, Naï P (2002). Concentration of some heavy metals in cattle reared in the vicinity of a metallurgic industry. Veterinarski Arhiv 72 (5): 259-267, 2002.


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Miranda, M, Lopez-Alonso M, Castillo C, Hernandez J, Benedito JL (2005). Effects of moderate pollution on toxic and trace metal levels in calves from a polluted area of Northern Spain. Environment International 31: 543-548. Moreiraa, FR, Borgesb TRM, Oliveira RM (2005). Comparison of two digestion procedures for the determination of lead in lichens by electrothermal atomic absorption spectrometry. Spectrochimica Acta Part B 60: 755– 758. Nemati, K, Bakar NKA, Abas MRB, Sobhanzadeh E, Low, KH (2010). Comparative study on open system digestion and microwave assisted digestion methods for metal determination in shrimp sludge compost. Journal of Hazardous Materials 182: 453–459. Neugebauer, EA, Sans Cartier GL, Wakeford BJ (2000). Methods for the Determination of Metals in Wildlife Tissues Using Various Atomic Absorption Spectrophotometry Techniques. Technical Report Series No. 337E. Canadian Wildlife Service, Headquarters, Hull, Québec, Canada. Nwude, DO, Okoye PAC, Babayemi JO (2010a). Blood heavy metal levels in cows at slaughter at Awka Abattoir, Nigeria. International Journal of Dairy Science 5(4): 264-270. Nwude, DO, Okoye PAC, Babayemi JO (2010b). Heavy metal levels in animal muscle tissue: a case study of Nigerian raised caatle. Research Journal of Applied Sciences 5(2): 146-150. Nwude, DO, Okoye PAC, Babayemi JO (2010c). Assessment of accumulation of heavy metals in the kidney of cattle as a function of seasonal variation. Africa Journal of Animal and Biomedical Sciences 5(3): 1-8. Nwude, DO, Okoye PAC, Babayemi JO (2011). Assessment of heavy metal concentrations in the liver of cattle at slaughter during three different seasons. Research Journal of Environmental Sciences 5(3): 288-294. Olowu, RA, Ayejuyo OO, Adejoroi A, Adewuyi GO, Osundiya MO, Onwordi CT, Yusuf KA, Owolabi MS (2010). Determination of Heavy Metals in Crab and Prawn in Ojo Rivers Lagos, Nigeria. E-Journal of Chemistry 7(2): 526-530. Raposo, Jr. JL, Oliveira SR, Nóbrega JA, Neto JAG (2008). Internal standardization and least-squares background correction in high-resolution continuum source flame atomic absorption spectrometry to eliminate interferences on determination of Pb in phosphoric acid. Spectrochimica Acta Part B 63: 992–995. Saavedraa, Y, Gonza´lezb A, Ferna´ndeza P, Blanco J (2004). A simple optimized microwave digestion method for multielement monitoring in musselsampl es. Spectrochimica Acta Part B 59: 533–541.

Tables Table 1: Observed filtrate colour in relation to the digestion conditions Sample Cow Digestion with HNO3 Temp. Time Colour of Filtrate (oC) (min) A 30 262 Light yellow Kidney A 30 262 Light yellow Liver A 30 262 Light yellow Muscle tissue B 30 262 Light yellow Kidney B 30 262 Light yellow Liver B 30 262 Light yellow Muscle tissue C 30 262 Light yellow Kidney Liver Muscle tissue Kidney Liver Muscle tissue Kidney Liver Muscle tissue

C C D D D E E E

30 30 30 30 30 30 30 30

262 262 245 245 245 245 245 245

Light yellow Light yellow Deep yellow Light yellow Light yellow Deep yellow Light yellow Deep yellow

Digestion with H3PO4 Temp. Time Colour of Filtrate (oC) (min) 186 35 Brown 186 35 Brown 186 35 Brown 186 35 Brown 186 35 Brown 186 35 Brown 186 35 Brown 186 186 171 171 171 171 171 171

35 35 33 33 33 33 33 33

Brown Brown Deep brown Brown Brown Deep brown Deep brown Brown


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Table 2: Levels of metals (mg/kg) in the different organs using the two different acids

HKAs = As in the kidney digested with HNO3, HLAs = As in the liver digested with HNO3, HMAs = As in the muscle tissue digested with HNO3, HKNi = Ni in the kidney digested with HNO3, HLNi = Ni in the liver digested with HNO3, HMNi = Ni in the muscle tissue digested with HNO3, HKCo = Co in the kidney digested with HNO3, HLCo = Co in the liver digested with HNO3, HMCo = Co in the muscle tissue digested with HNO3, HKPb = Pb in the kidney digested with HNO3, HLPb = Pb in the liver digested with HNO3, HMPb = Pb in the muscle tissue digested with HNO3, HKCd = Cd in the kidney digested with HNO3, HLCd = Cd in the liver digested with HNO3, HMCd = Cd in the muscle tissue digested with HNO3, PKAs = As in the kidney digested with H3PO4, PLAs = As in the liver digested with H3PO4, PMAs = As in the muscle tissue digested with H3PO4, PKNi = Ni in the kidney digested with H3PO4, PLNi = Ni in the liver digested with H3PO4, PMNi = Ni in the muscle tissue digested with H3PO4, PKCo = Co in the kidney digested with H3PO4, PLCo = Co in the liver digested with H3PO4, PMCo = Co in the muscle tissue digested with H3PO4, PKPb = Pb in the kidney digested with H3PO4, PLPb = Pb in the liver digested with H3PO4, PMPb = Pb in the muscle tissue digested with H3PO4, PKCd = Cd in the kidney digested with H3PO4, PLCd = Cd in the liver digested with H3PO4, PMCd = Cd in the muscle tissue digested with H3PO4.

Table 3: The Paired Samples Statistics Pairs Pair 1 Pair 2 Pair 3 Pair 4 Pair 5 Pair 6 Pair 7 Pair 8 Pair 9 Pair 10 Pair 11 Pair 12 Pair 13 Pair 14 Pair 15

HKAs PKAs HLAs PLAs HMAs PMAs HKNi PKNi HLNi PLNi HMNi PMNi HKCo PKCo HLCo PLCo HMCo PMCo HKPb PKPb HLPb PLPb HMPb PMPb HKCd PKCd HLCd PLCd HMCd PMCd

Mean 4.642 5.486 5.951 6.843 4.523 5.797 .094 .012 .108 .022 .054 .039 .010 .000 .023 .000 .029 .000 .620 .638 .674 .499 .721 .476 .086 .139 .057 .143 .069 .133

N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

Std. Deviation 1.599 3.082 .705 2.280 1.656 3.334 .083 .024 .078 .037 .058 .020 .020 .000 .025 .000 .042 .000 .344 .195 .098 .200 .379 .198 .031 .037 .012 .023 .028 .007

Std. Error Mean .715 1.378 .315 1.020 .741 1.491 .038 .011 .035 .016 .026 .009 .009 .000 .011 .000 .019 .000 .154 .087 .044 .089 .170 .088 .014 .016 .005 .010 .012 .003


Table 4: Levels of significance in the differences in levels of metals obtained using the two different acids for digestion Paired Samples Test Paired Differences T df Sig. 95% Confidence Interval (2-tailed) of the Difference Mean Std. Std. Lower Upper Deviatio Error n Mean Pair 1 HKAs – PKAs -.8444 3.8480 1.721 -5.6224 3.9336 -.491 4 .649 Pair 2 HLAs – PLAs -.8916 2.8664 1.281 -4.4507 2.6675 -.696 4 .525 Pair 3 HMAs – -1.274 4.6521 2.081 -7.0506 4.5022 -.612 4 .573 PMAs Pair 4 HKNi – PKNi .0800 .1016 .045 -.0462 .2062 1.760 4 .153 Pair 5 HLNi – PLNi .0856 .0771 .035 -.0101 .1813 2.481 4 .068 Pair 6 HMNi – PMNi .0146 .0679 .030 -.0697 .0989 .480 4 .656 Pair 7 HKCo – PKCo .0100 .0196 .009 -.0144 .0344 1.136 4 .319 Pair 8 HLCo – PLCo .0234 .0250 .011 -.0077 .0545 2.088 4 .105 Pair 9 HMCo – .0292 .0416 .019 -.0225 .0809 1.567 4 .192 PMCo Pair 10 HKPb – PKPb -.0178 .1634 .073 -.2207 .1851 -.244 4 .820 Pair 11 HLPb – PLPb .1748 .2500 .111 -.1356 .4852 1.563 4 .193 Pair 12 HMPb – .2458 .2722 .122 -.0921 .5837 2.019 4 .114 PMPb Pair 13 HKCd – PKCd -.0522 .0307 .014 -.0903 -.0140 -3.799 4 .019 Pair 14 HLCd – PLCd -.0860 .0322 .014 -.1260 -.0459 -5.962 4 .004 Pair 15 HMCd – -.0642 .0244 .011 -.0945 -.0338 -5.867 4 .004 PMCd

Figure 1: Comparison of the levels of the metals in various cow parts with regard to the digestion procedure

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